OPBSResearchMusculoskeletal
Must Know
- Humans carry 46 chromosomes (23 pairs). Each cell holds about 2 m of DNA.
- DNA uses four nucleotides. Guanine, cytosine, adenine and thymine.
- The genome is about 3 billion base pairs, sequenced in full in 2003.
- Only 2 to 3 percent of the genome codes for protein. The other 97 to 98 percent is noncoding.
- Every cell has the same DNA. Different cells differ because epigenetic marks switch genes on or off.
- Transcriptome is all the mRNA in a cell or tissue at a given time and state.
- Genotype plus epigenetics plus environment determine phenotype, the observable traits.
- Humans are diploid. Two identical alleles make a homozygote, two different alleles a heterozygote.
- Monogenic traits come from one gene. Polygenic traits come from two or more genes.
- Broad-sense heritability (H2) runs from 0 to 1. Higher values mean genes outweigh environment.
- A quantitative trait locus (QTL) is a DNA region linked to a trait, mapped using SNP markers.
Genetic Variation
| Variant | What it is | Share of genome |
|---|
| SNP | Change at one nucleotide position | Less than 1 percent |
| CNV | Gain or loss of a segment 1 kb or larger (deletion, insertion, duplication) | About 35 percent |
- Synonymous SNPs leave the protein unchanged. Nonsynonymous SNPs alter the amino acid sequence.
- SNPs outside coding regions can still alter splicing, transcription factor binding or mRNA breakdown.
- An SNP that alters gene expression is an eSNP (expression SNP).
- A single-base change in COL2A1 (type II procollagen) is linked to OA susceptibility.
Finding Disease Genes
| Method | Best for | Needs | Limit |
|---|
| Linkage analysis | Monogenic (Mendelian) disease | Large families over several affected generations | Poor for polygenic disease such as OA and osteoporosis |
| GWAS | Complex polygenic disease | Unrelated cases and controls | OA variants found have small effect sizes |
- GWAS rely on linkage disequilibrium mapping and give an unbiased screen for candidate genes.
More on OA association genes
- OA loci listed in the chapter include CHADL, DOT1L, TGFA, RUNX2, SMAD3, ALDH1A2 (hand), MCF2L and FTO.
- CHADL on 22q13.2 gave the largest hip odds ratio, 7.71.
- Most other OA variants have odds ratios close to 1.1 to 1.5.
Mouse Models and Genome Editing
| Term | Meaning |
|---|
| Transgenic | Any mouse with inserted DNA, from mouse or another species. Includes knock in and knockout |
| Global knockout | Existing gene inactivated by replacing or disrupting it |
| Knock-in | One-for-one swap of sequence at a locus, or insertion of new sequence |
| Conditional knockout | Floxed gene removed in chosen cells, usually by Cre-lox, and can be induced chemically |
| Tool | Year | Design time | Strength | Weakness |
|---|
| ZFNs | 1996 | Months | Fewer off-target mutations | Expensive, one site at a time |
| TALENs | 2010 | Weeks | Good specificity | Low efficiency, sensitive to DNA methylation, one site |
| CRISPR-Cas9 | 2012 | Days | High efficiency, many sites at once | More off-target effects |
More on knockout programmes
- By 2007 at least 11,000 mouse gene knockouts had been made.
- The Knockout Mouse Phenotyping Program aims at 20,000 genes, covering 90 percent of the mouse genome.
- A CRISPR mutant mouse is made by injecting guide RNA into a single cell zygote and transferring the blastocyst to a pseudo-pregnant female.
Gene Expression
- Transcription. RNA polymerase binds the promoter and makes an antiparallel complementary mRNA.
- Processing. Capping and polyadenylation aid transport to the ribosome. Splicing removes introns and joins exons.
- Alternative splicing. Lets one gene code for several proteins.
- Translation. Ribosome reads codons (nucleotide triplets). tRNA brings amino acids until a stop codon.
- Posttranslational modification. Covalent change after synthesis, such as phosphorylation, methylation, glycosylation, acetylation and ubiquitination.
- Activator transcription factors switch genes on. Repressors block RNA polymerase.
- Enhancers turn nearby genes on. Silencers turn them off.
- Musculoskeletal tissues are quiescent, with low expression until injury raises it (upregulation).
Measuring Expression
| Method | Principle | Point to remember |
|---|
| PCR | Primer driven enzymatic amplification | Primers 20 to 25 bp. Quantitative with fluorescent dye such as Sybr green |
| FISH | Probe hybridisation in situ | Single cell resolution, absolute mRNA quantification, shows localisation |
| Microarray | Hybridisation across the transcriptome | Discovery tool. Cross-hybridisation, poor at very low or high expression |
| RNA-seq | Next-generation sequencing | All RNA types, splice variants, gene fusions, exon-intron boundaries |
| scRNA-seq | Sequencing of individual cells | Defines cell subtypes and differentiation branch points |
- RNA is reverse transcribed to complementary DNA before most expression assays.
- Mass spectrometry is the most reliable protein quantification, sorting ions by mass-to-charge ratio.
More on data repositories
- Transcriptomic data go to the NIH Gene Expression Omnibus (GEO).
- Other repositories include ProteomicsDB and PRIDE (proteins), Roadmap Epigenomics, miRBase and MetaboLights.
- Pathway tools include DAVID, Ingenuity, PANTHER and MetaCore. The Gene Ontology consortium spans over 100 species.
- Northern blotting is now largely obsolete.
- Metabolomics studies small metabolites. Targeted work tests a hypothesis, untargeted work is discovery.
- Roadmap Epigenomics offers 111 reference epigenomes, each with about five histone marks.
Epigenetics
- Heritable change in gene expression across cell divisions with no change in DNA sequence.
- Epigenetic changes are potentially reversible, so suit drug therapy, already used in blood cancers.
- Methyl groups attach to cytosine in CpG sites, clustered in CpG islands.
- Promoter methylation silences genes. Demethylation of a promoter activates transcription.
- DNMT1, DNMT3A and DNMT3B methylate DNA. TET proteins remove methylation.
- Methyl-CpG-binding proteins recruit histone deacetylases, compacting chromatin into inactive heterochromatin.
- Dnmt3b regulates chondrogenic differentiation and is implicated in impaired fracture repair.
- Nucleosome core is DNA around a histone octamer, two each of H2A, H2B, H3 and H4.
- Histone acetylation activates transcription. Histone deacetylases silence it.
- H3K27 trimethylation represses transcription.
| Noncoding RNA | Size | Action |
|---|
| MicroRNA | 20 to 23 nucleotides, single stranded | Binds target mRNA, often the 3′ untranslated region, causing degradation or blocked translation |
| Long noncoding RNA | Over 200 nucleotides, no open reading frame over 100 amino acids | Chromatin modulation in the nucleus, scaffolding in the cytoplasm |
More on epigenetics in disease
- Diet, lifestyle, ageing, exercise, injury, smoking and diabetes can alter epigenetic marks.
- Genetics was founded by Gregor Mendel (1865) and epigenetics by Conrad Waddington (1942).
- Transcription factors need phosphorylation and partner factors to bind DNA.
- Cancer was the first disease linked to DNA methylation.
- Early musculoskeletal epigenetics came mainly from orthopaedic oncology.
- Epigenetic changes in chondrocytes from ageing and environmental risk factors may disturb cartilage homeostasis in OA.
- About 30,000 lncRNA transcripts exist in humans, forming most of the noncoding transcriptome. RNAcentral organises lncRNA databases.
Numbers to Remember
- Heritability. Rotator cuff tear 0.82, hip OA 0.60, knee OA 0.42, tendinopathy 0.40.
- OA due to genetics. 39 to 78 percent of cases.
- Coding genome. 2 to 3 percent.
- RNA products. About 85 percent of DNA yields lncRNA over 200 nucleotides.
- PCR. Copies rise exponentially, by hundreds of thousands.
Memory Aids
- Months, weeks, days. ZFN, TALEN, CRISPR in date order and in falling design time.
- Methylated means muted. Promoter methylation silences, acetylated histones open chromatin.
Exam Traps
- CNVs cover far more of the genome than SNPs, about 35 percent against under 1 percent.
- GWAS use unrelated cases and controls. Linkage analysis needs families.
- DNA methylation is reversible through TET proteins.
- Microarrays need no prior candidate genes. PCR targets a known sequence.
- CRISPR has more off-target effects than ZFNs.
- Osteocyte transcriptomes stay mostly osteoblast-like despite large expression changes.
Source
Rai MF, O’Keefe RJ, Brophy RH. Genetics, Gene Expression, and Epigenetics. In. Aaron RK, editor. Orthopaedic Basic Science. Foundations of Clinical Practice. 5th ed. Philadelphia, Wolters Kluwer, 2021.
Practise Type X questions on this topic
Reviewed by Professor Phong Tran, Head of Orthopaedic Surgery, Western Health. Last updated 17 October 2026.
